Monocyte derived macrophages (MDM) are the first cells infected with HIV-1 in vivo and act as a reservoir for infection of T cells. Cytokines are potent modulators of HIV, enhancing or inhibiting virus replication. Inasmuch as NK cells are the first line of defense against virus-infected cells and are known to produce a number of cytokines which modulate HIV (TNF, IFN-g, GM-CSF), we investigated the potential of NK cells to produce cytokines capable of preventing replication or spread of HIV by MDM. We found that fresh, purified NK cells treated with IL-2 and the NK cell lines, NK3.3 and YT, are a rich source of chemokines, including those which inhibit binding to the CCR-3 and CCR-5 chemokine co-receptors for HIV, and those with monocyte and T cell chemotactic/activating properties. These findings indicate that NK cells can produce multiple chemotactic factors capable of signalling through a wide variety of chemokine receptors. We also found that NK cells produce high levels of IL-10, as well as IL-13, a cytokine with potent monocyte differentiating properties and HIV inhibitory activity. Intriguingly, NK cells did not produce IL-4. Finally, we found that NK cells produce a unique factor(s) which prevents HIV-1 replication post-entry in MDM, but not T cells. Partial purification of this factor indicates that it is a small molecule of approximately 10 kD with a pI between 8.0 and 10.0. The small size and basic pI of this factor(s) were characteristic of a chemokine. However, the ability of this factor to inhibit virus replication, and not merely virus entry, indicated that the factor could not be a chemokine binding to the CCR-3 or CCR-5 chemokine co-receptors for HIV-1 present on MDM. Furthermore, pertusis toxin, a potent inhibitor of G-protein coupled receptor activity, had no effect on the inhibitory activity of this novel factor(s). Taken together, these data suggest that NK cells may play a much greater role in the regulation of HIV-1 expression in human MDM than previously thought possible. It is important to identify the panoply of cytokines involved and to delineate their potential to inhibit HIV replication in vitro and in vivo. Interferon-alpha (IFN-a) has potent anti-retroviral activity in several in vitro systems of both acute and chronic HIV-1 infection. Its clinical use has also proven beneficial in AIDS patients with relatively high numbers of CD4+ T cells. However, IFN-a species vary in their ability to inhibit virus replication in vitro, which may correlate with varying effects in vivo. Current studies are aimed at determining: 1) whether IFN-a components or recombinant hybrid species vary in their ability to inhibit acute HIV-1 infections of MDM and T cells; 2) whether anti-HIV activity is associated with expression of inducible nitric oxide synthase in MDM; 3) whether anti-HIV activity correlates with anti-proliferative activity; and 4) whether a species of IFN-a having low anti-proliferative activity and high anti-HIV activity can be identified, which may cause fewer toxic side effects when used clinically.